The use of monoclonal antibodies for cancer treatment has been quite successful. Antibody-drug conjugates have become potent new therapeutic options for the treatment of lymphoma and solid tumors, and immunoregulatory antibodies have recently demonstrated considerable success in clinical trials. The development of therapeutic antibodies is based on a deep understanding of cancer biology, protein engineering techniques, mechanisms of drug resistance, and the interaction between the immune system and cancer cells.
Antigens expressed on the surfaces of human cancer cells mean a broad range of targets that are overexpressed relative to normal tissues or mutated and selectively expressed. The key problem is to identify antigens appropriate for antibody-based therapies. These therapies mediate changes in ligand or receptor function (i.e., function as agonists or antagonists), regulate the immune system by antibody-dependent cell cytotoxicity (ADCC), and deliver a specific drug bound to a specific antibody that targets a specific antigen, thereby exhibiting their efficacy. Molecular techniques that can change antibody pharmacokinetics, activity, function, size and immunostimulatory activity have emerged as key elements in the development of new antibody-based therapies. Evidence from clinical trials of therapeutic antibodies directed against cancer patients emphasizes the importance of the binding affinities of antibodies for target antigens, the selection of antibody structures, and approaches for the selection of optimized antibodies, including therapeutic approaches (signaling inhibition or immune function).
In connection with this, studies on antibodies against epidermal growth factor receptor (EGFR) antigen have been conducted. The EGFR is the 170 kilodalton membrane lipoprotein product of the proto-oncogene c-erb B. The sequence of the EGFR gene is known. The EGFR gene is the cellular homolog of the erb-B oncogene originally identified in avian erythroblastasis viruses. Activation of this oncogene by gene amplification has been observed in a variety of human tumors.
EGFR is overexpressed on various types of human solid tumors. EGFR overexpression has been observed in certain lung, breast, colon, gastric, brain, bladder, head and neck, ovarian, kidney and prostate carcinomas. Both epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) have been demonstrated to bind to EGFR and to lead to cellular proliferation and tumor growth. In addition, amplification, point mutations and splice variants of EGFR have been reported in several human cancers.
EGFR variants are caused by gene rearrangement accompanied by EGFR gene amplification. There are eight major variants of EGFR that are known: (i) EGFRvI lacks a majority of the extracellular domain of EGFR, (ii) EGFRvII consists of an 83 aa in-frame deletion in the extracellular domain of EGFR, (iii) EGFRvIII consists of a 267 aa in-frame deletion in the extracellular domain of EGFR, (iv) EGFRvIV contains deletions in the cytoplasmic domain of EGFR, (v) EGFRvV contains deletions in cytoplasmic domain of EGFR, (vi) EGFR.TDM/2-7 contains a duplication of exons 2-7 in the extracellular domain of EGFR, (vii) EGFR.TDM/18-25 contains a duplication of exons 18-26 in the extracellular domain of EGFR, and (viii) EGFR.TDM/18-26 contains a duplication of exons 18-26 in the tyrosine kinase domain of EGFR. In addition, there is a second, more rare, EGFRvIII mutant (EGFRvIII/AΔ12-13) that possesses a second deletion that introduces a novel histidine residue at the junction of exons 11 and 14.
EGFRvIII is the most commonly occurring variant of the epidermal growth factor (EGF) receptor in human cancers, and is expressed in about 30% of glioblastoma multiforme (GBM) patients, but is not expressed in normal tissue. This variant of the EGF receptor contributes to tumor progression through constitutive signaling in a ligand independent manner.
Mutations or rearrangements in genes that potentially drive neoplasia can be identified in many cancers. Results have shown that oncogenic proteins can contribute to cancer stem cell-related pathways. It stands to reason that the products of such altered genes could be used to identify and potentially target cancer stem cells. In practice, this approach has been difficult to establish because driver mutations are present in cells throughout the mass and typically are not specific to any subpopulation. Thus, mutant proteins may not have any direct role in cancer stem cells and generally potentiate tumor growth. In addition, most altered proteins are intracellular.
The correlation between mutant proteins and cancer stem cells is not clear. Glioblastoma tumors are known to frequently express EGFRvIII, an EGFR variant expressed through gene rearrangement and amplification. Since tyrosine phosphorylation sites are always present in an activated form, they show strong tumorigenicity. However, despite this modification, the expression of EGFRvIII is limited. In cancer stem cells, EGFRvIII is highly expressed with CD133, and EGFRvIII+/CD133+ cells have high regeneration and tumor initiation capability. EGFRvIII+ cells were associated with stem/precursor markers, whereas differentiation markers were found in EGFRvIII− cells. Expression of EGFRvIII was lost in normal cell culture, but maintained in tumor sphere culture. In addition, cultured cells simultaneously express EGFRvIII+/CD133+, and are regenerated, and have tumor-initiating ability.
In order to treat cancer that overexpresses EGFRvIII, an anti-EGFRvIII antibody is required which is capable of binding to EGFRvIII with high affinity and inhibiting the growth of cancer cells.
Antibody therapeutic agents such as Cetuximab, which bind specifically to EGFR, have been developed conventionally. However, these antibody therapeutic agents entail problems that antigen specificity for EGFRvIII mutant cancer cells is very low, and that the inhibition of cancer cell growth does not appear.
Under this technical background, the present inventors have made extensive efforts to develop an anticancer therapeutic antibody that binds specifically to EGFRvIII. As a result, the present inventors have developed an anti-EGFRvIII antibody, which binds to EGFRvIII with high affinity, by using phage display technology, and have found that this anti-EGFRvIII antibody can significantly inhibit migration of cancer cells, thereby completing the present invention.